|Molecular Vision 2007;
Received 12 September 2006 | Accepted 19 June 2007 | Published 21 June 2007
A novel mutation of the Keratin 12 gene responsible for a severe phenotype of Meesmann's corneal dystrophy
Lori S. Sullivan,3
Eric B. Baylin,2 Ramon Font,4
Stephen P. Daiger,3
Jay S. Pepose,5
Thomas E. Clinch,6
Xinping C. Zhao,2
Richard W. Yee1,2
1Hermann Eye Center and 2Department of Ophthalmology and Visual Science, The University of Texas Health Science Center at Houston, Houston, Texas; 3School of Public Health, The University of Texas Health Science Center at Houston, Houston, Texas; 4The Cullen Eye Institute, Baylor College of Medicine, Houston, Texas; 5Department of Ophthalmology and Visual Sciences, Washington University Medical Center, St. Louis, MO; 6John A. Moran Eye Center, University of Utah Health Sciences Center, Salt Lake City, UT
Correspondence to: Richard W. Yee, M.D., Joe M. Green Chair in Ophthalmology, The Hermann Eye Center, University of Texas Health Science Center at Houston, 6411 Fannin, Houston, TX, 77030; Phone: (713) 704-1711; FAX: (713) 704-9002 email: email@example.com
Purpose: To determine if a mutation within the coding region of the keratin 12 gene (KRT12) is responsible for a severe form of Meesmann's corneal dystrophy.
Methods: A family with clinically identified Meesmann's corneal dystrophy was recruited and studied. Electron microscopy was performed on scrapings of corneal epithelial cells from the proband. Mutations in the KRT12 gene were sought using direct genomic sequencing of leukocyte DNA from two affected and two unaffected family members. Subsequently, the observed mutation was screened in all available family members using polymerase chain reaction and direct sequencing.
Results: A heterozygous missense mutation (Arg430Pro) was found in exon 6 of KRT12 in all 14 affected individuals studied. Unaffected family members and 100 normal controls were negative for this mutation.
Conclusions: We have identified a novel mutation in the KRT12 gene that is associated with a symptomatic phenotype of Meesmann's corneal dystrophy. This mutation results in a substitution of proline for arginine in the helix termination motif that may disrupt the normal helix, leading to a dramatic structural change of the keratin 12 protein.
Meesmann's corneal dystrophy (MCD), a bilaterally symmetric disorder, is characterized by fragility of the corneal epithelium [1,2]. MCD exhibits myriads of fine punctate opacities and cysts in the epithelium and occasionally in Bowman membrane and can be diagnosed on slit-lamp examination. While early disease is often asymptomatic, multiplication of the cysts throughout late adolescence and adulthood can cause epithelial erosion, leading to lacrimation, photophobia, and pain. In most cases, visual acuity is rarely affected; however, subepithelial scarring can occasionally produce irregular astigmatism and irrevocable visual deficits. Light microscopy often shows a thickened basement membrane and electron microscopy will reveal affected corneal epithelial cells with highly characteristic intracytoplasmic cysts containing an electron dense material referred to as "peculiar" substance [2,3].
The structural integrity of corneal epithelial cells derives largely from its composition of cytoskeletal intermediate filaments, specifically keratin 3 and keratin 12 [4,5]. These molecules function as heterodimers of type I and type II keratin , whereby disruption of a single keratin polymer can compromise epithelial structure and function . Until the present study, 14 mutations in KRT12 and two mutations in KRT3 had been found in MCD patients. All mutations were located in either helix-initiation or helix-termination motif [8-17]. The motifs were highly conserved among keratin proteins, and the domains are important in intermediate filament assembly and are likely involved in mediating end-to-end interactions between keratin heterodimers. Interestingly, there were more mutations reported in the helix-initiation motif than in the helix-termination motif of these two keratin genes. For example, 10 different mutations have been identified thus far in the helix-initiation motif of KRT12, including substitution of amino acid M129T , Q130P , N133K , R135S , R135G , R135I , R135T , A137P , L140R , and V143L . However, there have been only four mutations reported in the helix-termination motif in KRT12: I426V , I426S , Y429D , and Y429C . Also, the two reported mutations in KRT3 (R503P , E509K ) are in the helix-termination motif and not the helix-initiation motif.
In this paper, we report a new case of MCD found in a family who exhibited severe spectrum of disease. We performed mutation screening on the family and discovered a novel missense mutation in the helix-termination motif of KRT12.
This study was approved by the institution review board of the University of Texas Health Science Center at Houston. All patients were informed about the study and signed a consent form in accordance with guidelines set forth by the Declaration of Helsinki. A large family with 17 members affected with Meesmann's corneal dystrophy was identified and given complete eye exams. For detailed examination of patient corneas, bilateral corneal scrapings were performed on the proband, and scrapings of corneal epithelial cells were subsequently fixed in 2% glutaraldehyde and visualized by using conventional electron microscopy.
Detection of KRT12 mutations
Genomic DNA was extracted from peripheral blood obtained from all family members (Figure 1). Mutation screening of the KRT12 gene was done initially in one affected and one unaffected family member. The entire coding region (all eight exons) was examined by direct polymerase chain reaction (PCR) sequencing. The primers used for amplification of all exons of KRT12 are shown in Table 1. PCR products were examined on 2% agarose gels and then sequenced using an AB 3100 genetic analyzer (Applied Biosystems, Foster City, CA). A mutation in exon 6 was confirmed by using the restriction enzyme FauI (New England Biolabs, Beverly, MA), according to the manufacturer's protocol, to digest PCR products from the proband and his unaffected sister. Digested samples were run on 2.5% agarose gels, and the bands were visualized by ethidium bromide staining. Also, DNA samples from the remaining family members were PCR amplified and sequenced for exon 6 of KRT12. The mutation was further verified by sequencing of PCR products amplified from 100 Centre d'Etude du Polymorphisme Humain (CEPH) control samples.
The proband was a 33-year-old male with symptomatic MCD from birth. He presented with symptoms of photophobia, lacrimation, periodic burning, and irritation. He had flare-ups almost monthly that lasted from two days to one week, followed by remission. Unlike most patients with MCD, his visual acuity was significantly impaired (20/200 visual acuity score), and he wore corrective lenses. Slit-lamp examination disclosed dense microcysts in the visual axis limited to the anterior epithelium in addition to significant anterior basement membrane scarring (Figure 2). Clinical histories obtained from family members were remarkably similar. The general impression by collective slit-lamp examinations was consistent with MCD. In this family, however, MCD presents an unusually severe spectrum of disease.
Electron microscopy analysis of the proband showed intraepithelial cysts involving the superficial cell layers of the cornea. The cysts were bordered by microvillous processes, indicating they were formed by acantholysis (Figure 3). The cysts contained a "peculiar" substance intermixed with numerous round vacuoles that were surrounding electron-dense elongated filamentous material .
Molecular Genetic Analysis
Examination of phenotypes within this five-generation family indicated an autosomal dominant mode of inheritance (Figure 1). Since a majority of MCD patients have mutations in KRT12, we hypothesized that a mutation in KRT12 might be associated with the phenotype observed in this family. To test this hypothesis, we sequenced the entire coding region of KRT12 in selected family members. PCR analysis and direct DNA sequencing were performed in a screening panel of two affected and two unaffected individuals to determine if there was a mutation within the coding region of the KRT12 gene that is associated with the form of MCD found in this family. This analysis identified a novel heterozygous missense mutation (Arg430Pro; CGC>CCC) in exon 6 of the KRT12 gene in the affected individuals but not in the unaffected normal controls of the family (Figure 4A). Substitution of C for G in codon 430 resulted in the gain of a recognition site for the restriction enzyme FauI. Since there is another FauI site 30 bp apart from the site, digestion of the exon 6 PCR products from patients with this mutation produced bands of 220, 81, and 30 bp, while normal alleles produced bands of 220 and 111 bp (Figure 4B). As expected, FauI digestion of PCR products generated a 81 bp fragment from the proband but not from his unaffected sister. Therefore, substitution of C for G in codon 430 is likely associated with MCD in this family. To determine whether this mutation was cosegregated with the disease, we examined and compared the sequence of exon 6 in all DNA samples obtained from the remaining family members. Sequence analysis showed that the affected individuals had this mutation but the unaffected individuals did not (data not shown). To further determine if this nucleotide substitution were associated with the disease phenotype, we performed sequence analysis of exon 6 of KRT12 on the 100 CEPH control individuals. Our goal was to rule out the possibility that this alternation in DNA was a rare single nucleotide polymorphism occurring in the control population. Direct sequencing of exon 6 of the KRT12 gene from these additional individuals failed to find the same sequence variation. Therefore, the Arg430Pro mutation that was identified and was cosegregated with the MCD in this family may be responsible for MCD of this family.
The family members we examined exhibited an unusually severe phenotype for MCD characterized by early onset of the disease, ocular irritation, and poor visual acuity. As an example, the proband has undergone four phototherapeutic keratectomy procedures for alleviation of recurrent symptoms. Irregular astigmatism has been problematic despite the improvement of the recurrent corneal erosions. This rather atypical presentation may reflect the unique Arg430Pro missense mutation, which lies within the helix termination motif of the α-helical rod domain of KRT12 (Figure 4B). The helix-termination motif is highly conserved among keratin proteins and is critical for keratin fiber formation. In addition to the helix initiation motif, mutations in this helix-termination motif are known to result in more severe phenotypes of other inherited epidermal diseases [18-22]. It is noteworthy that most documented keratin mutations responsible for MCD are in the helix initiation motif [9-12,14], and a few families have been identified with a mutation in the helix termination region [11,13,15,16]. It is possible that the clinical severity observed in our pedigree is due to the structural change of the keratin 12 protein caused by the replacement of arginine with a proline residue, which is particularly incompatible with an α-helical structure. The amino group of proline imposes both geometric and electrostatic constraints on the α-helix by elimination of N-Cα bond rotation and available hydrogen bonds. In contrast, a recent article described an essentially asymptomatic family with clinically diagnosed MCD caused by a different KRT12 mutation. In this case, the identified KRT12 mutation resulted from a substitution of a nonpolar for another nonpolar amino acid, probably causing a less severe disruption of the keratin protein structure (and a less severe phenotype) .
Unlike other mutations, the Arg430Pro leads to a replacement of a highly charged amino acid with a hydrophobic amino acid. We believe that the helix-termination motif in the mutant keratin protein may be no longer functional for the fiber formation. We are unaware of any other reports of KRT12 mutations in MCD with the added instability of a proline substitution in the helix terminal motif. Our search of Medline, the Human Gene Mutation Database, or OMIM turned up no such mutation, although one group reported a proline substitution in the helix terminal motif in KRT3 . We postulate that the severity of MCD in our pedigree is related specifically to the biochemical significance of the novel Arg430Pro mutation described herein.
This work was supported in part by grants from the Hermann Eye Fund, Houston, TX, a departmental core grant EY10608, grant K08 EY00350 from the National Eye Institute (R.W.Y.), and grants from the Retina Research Foundation, Houston TX, and Research to Prevent Blindness, Inc., New York, New York; the Department of Ophthalmology and Visual Science, University of Texas Health Science Center at Houston, and Dr. Font.
1. Meesmann A, Wilke F. Klinsche und anatomische Untersuchungen uber eine bisher unbekannte, dominant verebre Epitjeldystrophie der Hornhaut. Klin Monatsbl 1939; 103:361-91.
2. Tremblay M, Dube I. Meesmann's corneal dystrophy: ultrastructural features. Can J Ophthalmol 1982; 17:24-8.
3. Fine BS, Yanoff M, Pitts E, Slaughter FD. Meesmann's epithelial dystrophy of the cornea. Am J Ophthalmol 1977; 83:633-42.
4. Rodrigues M, Ben-Zvi A, Krachmer J, Schermer A, Sun TT. Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium. Differentiation 1987; 34:60-7.
5. Chaloin-Dufau C, Sun TT, Dhouailly D. Appearance of the keratin pair K3/K12 during embryonic and adult corneal epithelial differentiation in the chick and in the rabbit. Cell Differ Dev 1990; 32:97-108.
6. Hatzfeld M, Weber K. The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression. J Cell Biol 1990; 110:1199-210.
7. Kao WW, Liu CY, Converse RL, Shiraishi A, Kao CW, Ishizaki M, Doetschman T, Duffy J. Keratin 12-deficient mice have fragile corneal epithelia. Invest Ophthalmol Vis Sci 1996; 37:2572-84.
8. Corden LD, Swensson O, Swensson B, Smith FJ, Rochels R, Uitto J, McLEAN WH. Molecular genetics of Meesmann's corneal dystrophy: ancestral and novel mutations in keratin 12 (K12) and complete sequence of the human KRT12 gene. Exp Eye Res 2000; 70:41-9.
9. Corden LD, Swensson O, Swensson B, Rochels R, Wannke B, Thiel HJ, McLean WH. A novel keratin 12 mutation in a German kindred with Meesmann's corneal dystrophy. Br J Ophthalmol 2000; 84:527-30.
10. Irvine AD, Coleman CM, Moore JE, Swensson O, Morgan SJ, McCarthy JH, Smith FJ, Black GC, McLean WH. A novel mutation in KRT12 associated with Meesmann's epithelial corneal dystrophy. Br J Ophthalmol 2002; 86:729-32.
11. Yoon MK, Warren JF, Holsclaw DS, Gritz DC, Margolis TP. A novel arginine substitution mutation in 1A domain and a novel 27 bp insertion mutation in 2B domain of keratin 12 gene associated with Meesmann's corneal dystrophy. Br J Ophthalmol 2004; 88:752-6.
12. Nishida K, Honma Y, Dota A, Kawasaki S, Adachi W, Nakamura T, Quantock AJ, Hosotani H, Yamamoto S, Okada M, Shimomura Y, Kinoshita S. Isolation and chromosomal localization of a cornea-specific human keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy. Am J Hum Genet 1997; 61:1268-75.
13. Irvine AD, Corden LD, Swensson O, Swensson B, Moore JE, Frazer DG, Smith FJ, Knowlton RG, Christophers E, Rochels R, Uitto J, McLean WH. Mutations in cornea-specific keratin K3 or K12 genes cause Meesmann's corneal dystrophy. Nat Genet 1997; 16:184-7.
14. Takahashi K, Takahashi K, Murakami A, Okisaka S, Kimura T, Kanai A. Heterozygous Ala137Pro mutation in keratin 12 gene found in Japanese with Meesmann's corneal dystrophy. Jpn J Ophthalmol 2002; 46:673-4.
15. Coleman CM, Hannush S, Covello SP, Smith FJ, Uitto J, McLean WH. A novel mutation in the helix termination motif of keratin K12 in a US family with Meesmann corneal dystrophy. Am J Ophthalmol 1999; 128:687-91.
16. Nichini O, Manzi V, Munier FL, Schorderet DF. Meesmann corneal dystrophy (MECD): report of 2 families and a novel mutation in the cornea specific keratin 12 (KRT12) gene. Ophthalmic Genet 2005; 26:169-73.
17. Chen YT, Tseng SH, Chao SC. Novel mutations in the helix termination motif of keratin 3 and keratin 12 in 2 Taiwanese families with Meesmann corneal dystrophy. Cornea 2005; 24:928-32.
18. Coulombe PA, Hutton ME, Letai A, Hebert A, Paller AS, Fuchs E. Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. Cell 1991; 66:1301-11.
19. Lane EB, Rugg EL, Navsaria H, Leigh IM, Heagerty AH, Ishida-Yamamoto A, Eady RA. A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering. Nature 1992; 356:244-6.
20. Chen MA, Bonifas JM, Matsumura K, Blumenfeld A, Epstein EH Jr. A novel three-nucleotide deletion in the helix 2B region of keratin 14 in epidermolysis bullosa simplex: delta E375. Hum Mol Genet 1993; 2:1971-2.
21. Cheng J, Syder AJ, Yu QC, Letai A, Paller AS, Fuchs E. The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes. Cell 1992; 70:811-9.
22. Bonifas JM, Rothman AL, Epstein EH Jr. Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities. Science 1991; 254:1202-5.